The invention relates to the scanning and representation of daughter ion spectra for the purpose of library searching and determining the structural characteristics of parent ions in ion traps.
Paul ion traps consist of a ring electrode supplied with high frequency and two end cap electrodes; ions can be stored inside. The ion traps can be used as mass spectrometers by ejecting the stored ions mass-selectively and scanning them with a secondary-electron multiplier. There are several different methods known for ion ejection which will not be discussed in further detail here.
Ion cyclotron resonance mass spectrometers are a different type of ion traps in which the ions can be stored in a high-constancy magnetic field and in additional electrical fields. After excitation the circular movements of the ions can be used to measure the ratio between their masses and their charges.
Ion trap mass spectrometers have special features which make their use in many types of analyses useful. In particular, selected ion types (so-called xe2x80x9cparent ionsxe2x80x9d) can be xe2x80x9cisolatedxe2x80x9d in the ion trap (freed from all other ion types and stored alone) and fragmented with the help of a damping or collision gas by molecular collisions after excitation of their oscillating movements. This method is called xe2x80x9ctandem in timexe2x80x9d. In high frequency quadrupole ion traps, for example, excitation occurs by applying alternating voltage of an appropriate frequency to the end caps. The spectra of these ions generated by fragmentation are known as the xe2x80x9cdaughter ion spectraxe2x80x9d of the associated parent ions. xe2x80x9cGranddaughter spectraxe2x80x9d can also be scanned as fragment ion spectra of a selected daughter ion. In favorable cases, it is possible to scan such fragmentation spectra through to the tenth generation or more.
In contrast to other types of tandem mass spectrometry, particularly to xe2x80x9ctandem in spacexe2x80x9d mass spectrometry using two mass spectrometers in series, the daughter spectra in ion traps are relatively simple for the most part. Often a daughter ion spectrum contains only a very few different types of daughter ions. The reason for this is thatxe2x80x94particularly in a high frequency Paul ion trapxe2x80x94only the parent ions receive energy through resonance excitation, but none of the other ions accidentally found in the trap. Particularly, no further energy is fed to the resulting daughter ions; on the contrary, the daughter ions are cooled immediately after their formation by the ambient damping gas and can therefore usually not decompose any further. Therefore such a daughter ion spectrum usually contains relatively little information regarding the structure of the parent ion. Only in special cases are larger numbers of daughter ions formed; the requirement is that there are many equally loose binding points with almost equally low binding energies in rather large molecules. In fact, this case only occurs among larger biopolymers.
On the other hand, it is also possible to re-isolate and to fragment daughter ions in ion trap mass spectrometers and thus measure the fragment ions over several isolation and fragmentation generations. This is sometimes called MSn. Although every single one of the spectra so measured provides only little information regarding the structure of the original ion, the entirety of all fragment ions does contain a large amount of structural information, particularly due to the fact that the relationships between the fragment ions are known. However, it is laborious to pick out information from a possibly enormous number of individual spectra.
In xe2x80x9ctandem in spacexe2x80x9d mass spectrometers, daughter ion spectra are scanned in an entirely different way. Parent ions selected by the first mass spectrometer are.injected with considerable kinetic energy into a chamber with collision gas in front of the second spectrometer. About every impact transfers so much energy to the ion that it fragments. The daughters continue to fly at a barely reduced velocity and are simultaneously subjected to further collision processes with fragmentations. In this way, a xe2x80x9cdaughter ion spectrumxe2x80x9d is produced which is a mixed spectrum of actual daughter ions mixed with granddaughter ions and great-granddaughter ions from several generations and several branches. Actually, this mixed spectrum contains a great deal of structural information. However, this information is also difficult to extract, this time due to the fact that the relationships between the fragment ions are unknown. It is no longer possible to tell from a fragment ion whether it was formed directly from the parent ion, indirectly from another fragment ion or in an even more complicated way. In the classical way of mass spectrometry, tedious experiments with isotopically marked molecules were necessary to get the wanted structural information.
Objective of the invention
It is the objective of the invention to find a technique to ease library searches in daughter, granddaughter and great-granddaughter spectra for the identification of a substance, and to facilitate the extraction of structural information on parent ions in an easier way then to look through a large number of fragment ion spectra.
It is the basic idea of the invention to display the various ion types which are scanned in the fragment ion spectra of various isolation and fragmentation generations, in one single common xe2x80x9ccombined descendant spectrumxe2x80x9d of a progenitor ion. In this way, a spectrum is produced which corresponds far more closely to a daughter spectrum from other types of tandem mass spectrometry, at least with respect to the masses appearing in the spectrum. Libraries of MS/MS spectra obtained with tandem-in-space mass spectrometers, can be used to identify substances. Most of the search programs are written in such a manner that utmost weight is put on the appearance of masses, not on their intensities. Even if there are no MS/MS libraries for tandem-in-space spectra, library searches are much easier in libraries with combined descendant spectra than with a bundle of related MSn spectra.
For the intensity ratios of individual fragment ion types from various origins among each other, special conventions must be made, although the display primarily serves the purpose of structural determination and library identification of the progenitor ion type thus analyzed, and in these cases the intensities are generally less important.
The combined descendant spectrum can be either presented graphically, or as a list with masses and intensities of the ion types. In the former case the intensities of ion types are usually plotted as vertical lines on a horizontal mass scale (xe2x80x9cbar graph spectrumxe2x80x9d).
It is a further idea of the invention to designate the relationships among the ions in the spectrum. For example, in a spectrum list the mass differences from the direct parent ion (predecessor ion) can be entered in a third column in addition to masses and intensities. In a graphically displayed bar graph spectrum, the relationships can be incorporated in the same way as a design drawing is dimensioned, and mass differences can also be indicated. The quantity and quality of information in such spectra far surpasses that of daughter spectra from other types of tandem mass spectrometry. The mass differences of the relationships can also be used for improving library searches, e.g. by consideration of these differences in a matching score which characterizes the quality of a match.
In a further configuration of the invention, the names or abbreviations of lost neutral fragments can be directly plotted into the spectrum instead of the mass differences. Particularly in the case of biopolymers such as proteins or oligonucleotides, the structure is made up of a linear chain of entities (building blocks) and the ions are fragmented following this pattern according to relatively simple rules. The masses of the entities are known precisely: for proteins it is the masses of the twenty amino acids, for oligonucleotides of the four nucleotides (bases).
Using a list with the dimensions and names of these entities, series of losses of entities in the descendants spectrum can be identified directly.
In the combined descendants spectrum, either all measured ion types from the fragment ion spectra can be plotted, or only selected ion types. For purposes of convenience, selection is conducted according to previously specified criteria which can be defined, for example, via intensity thresholds or via mass thresholds of mass differences from the predecessor ion. In the latter case, biopolymer sequences can be made obvious with particular ease.
Rather than selecting ion types according to the criteria at the time the combined descendants spectrum is formed, it can also be done already at the time of measurement. For example, with appropriately defined criteria, only the fragment ion spectra of those fragment ions need be scanned which have a mass interval from their predecessor ion beneath a threshold. Automatic selection at this point in time permits automatic scanning of all these fragment ion spectra.